Antiviral Therapy 2009 14: 965–974 (doi: 10.3851/IMP1399) Original article P-glycoprotein, multidrug resistance-associated proteins and human organic anion transporting polypeptide influence the intracellular accumulation of atazanavir Omar Janneh1,2*, Tariq Anwar3, Christof Jungbauer 4, Stefan Kopp5, Saye H Khoo2, David J Back2 and Peter Chiba5 1Department of Biomolecular and Sport Sciences, Coventry University, Coventry, UK 2Department of Pharmacology, School of Biomedical Sciences, University of Liverpool, Liverpool, UK 3School of Biomedical Sciences, University of Ulster, County Londonderry, UK 4Austrian Red Cross, Blood Donation Centre for Vienna, Lower Austria and Burgenland, Vienna, Austria 5Institute of Medical Chemistry, University of Vienna, Vienna, Austria *Corresponding author: e-mail: [email protected] Background: Drug efflux (for example, P-glycoprotein HPIs on uptake were also evaluated. Data from digitonin [P-gp], multidrug resistance-associated proteins [MRPs] permeabilized cells allowed the evaluation of the contri- and breast cancer resistance protein [BCRP]) and influx bution of cellular binding to total drug uptake, whereas (for example, human organic anion transporting polypep- the inhibitory effect of ATV on P-gp was assessed by tide [hOCTP] or human organic anion transporting daunomycin efflux/uptake assays. polypeptide [hOATP]) transporters alter the cellular con- Results: [3H]-ATV is lipophilic and accumulates in the centrations of some HIV protease inhibitors (HPIs). Here, cultured cells as follows: CEM>CEME1000>CEMVBL. Tariq- we studied the lipophilicity and uptake of [3H]-atazanavir uidar, GF120918 and daidzein significantly increased the 3 (ATV) in CEM (parental), CEMVBL (P-gp-overexpressing), uptake of [ H]-ATV in the cultured cells. By contrast, only CEME1000 (MRP1-overexpressing) and peripheral blood daidzein and tipranavir significantly increased uptake in mononuclear cells (PBMCs), and evaluate the effects of PBMCs, with tariquidar and frusemide devoid of effects, modulators of drug transporters on uptake. whereas dipyridamole, E-3-S, GPV031 and genistein sig- Methods: Lipophilicity was measured by octanol/saline nificantly decreased accumulation. ATV inhibits P-gp partition method. The influence of influx/efflux trans- activity; manipulation of uptake with digitonin suggests porters on uptake was evaluated in the absence and binding of [3H]-ATV to P-gp. presence of inhibitors of P-gp (GPV031), P-gp/BCRP Conclusions: [3H]-ATV is lipophilic, a P-gp, MRP and (tariquidar and GF120918), P-gp/MRP1 (dipyrida- hOATP substrate and an inhibitor of P-gp. Concomitant mole and daidzein), MRP1/2 (frusemide and genistein), administration of ATV with drugs and dietary components hOATP/hOCTP (estrone-3-sulfate [E-3-S]) and hOATP/ (for example, daidzein and genistein) that interact with hOCTP/MRP (probenecid). The effects of a number of these transporters could alter its pharmacokinetics. Introduction HIV protease inhibitors (HPIs) in combination with resistance-associated proteins (MRPs) and breast can- other antiretrovirals (for example, reverse tran- cer resistance protein (BCRP) [1–3]. In addition, there scriptase inhibitors) play a pivotal role in the man- are some known anionic and cationic transporters, agement of HIV-infected patients. The therapeutic such as human organic anion transporting polypep- effects of these drugs is dependent on their cellular tide (hOATP) and human organic cation transport- accumulation, which is in part affected by the expres- ers (hOCTs), respectively, which mediate the influx/ sion of ATP binding cassette family of drug efflux elimination of many clinically important drugs and transporters, such as P- glycoprotein (P-gp), multidrug xenobiotics, and these have been shown to limit the ©2009 International Medical Press 1359-6535 (print) 2040-2058 (online) 965 AVT-09-OA-1243_Janneh.indd 965 30/10/09 15:14:25 O Janneh et al. accumulation of some HPIs [2,4–6]. All these trans- Methods porters are differentially expressed in the liver, kidney, circulating lymphocytes, brain, testes and intestines – Reagents sites important in drug disposition, or which might act [3H]-ATV and [14C]-mannitol (specific activities 3.1 Ci/ as sanctuary for the replicating virus and/or support mmol and 50 mCi/mmol, respectively) were purchased viral replication. Clearly, low intracellular/plasma from Moravek Biochemicals and Radiochemicals (Brea, drug concentration or altered distribution within these CA, USA). RTV and lopinavir (LPV) were obtained sites could affect clinical outcome. from Abbott Laboratories (North Chicago, IL, USA); The transporters work in concert to control the bio- nelfinavir (NFV), indinavir (IDV), SQV, amprenavir availability of clinically important drugs and endog- (APV), ATV and tipranavir (TPV) were obtained from enous compounds. Thus, together with the cytochrome Agouron Pharmaceuticals (San Diego, CA, USA), Merck P450 enzymes, these transporters are important deter- (Rahway, NJ, USA), Roche (Welwyn Garden City, UK), minants of drug–drug interactions, drug efficacy and Glaxo Wellcome (Middlesex, UK), Bristol–Myers Squibb toxicity [7]. Indeed, previous studies have shown (Hounslow, UK) and Boehringer Ingelheim (Berkshire, the oral availability, brain, and fetal penetration of UK), respectively. Tariquidar was donated by Xenova saquinavir (SQV) to be limited by P-gp [8,9]. Simi- Group Plc (Berkshire, UK). The peripheral blood mono- larly, the intracellular accumulations of a number of nuclear cells (PBMCs) were isolated from blood buffy HPIs have been shown to be limited by P-gp, MRP1, coats, which were obtained from the Regional Blood MRP2 and BCRP [1,2,8–19]. There is ample evidence Transfusion Centre (Liverpool, UK). CEM (parental), that some HPIs also induce and inhibit the expression CEMVBL (VBL) and CEME1000 (E1000) cell lines were of drug efflux transporters, such as P-gp, MRP1/2 and from R Davey (Bill Walsh Cancer Research Laborato- BCRP [12,16,20–25]. However, the role of BCRP in ries, Royal North Shore Hospital, Sydney, Australia). the efflux of HPIs is equivocal [1,3,24–27]; there is Lymphoprep was purchased from Axis Shields (Oslo, evidence that BCRP induces resistance to nucleoside Norway). GPV031 was a gift from G Ecker (Depart- reverse transcriptase inhibitors [27]. ment of Medicinal Chemistry, University of Vienna, We have shown that the intracellular concentrations Vienna, Austria). All other chemicals were supplied by of HPIs are amenable to manipulation by coadministra- Sigma Chemical Co. (Poole, UK). tion with inhibitors of drug efflux transporters [1,2,13]. A potential use of such agents would be to enhance intes- Cell culture tinal drug absorption and increase drug penetration to The cultured cells used in this study were CEM (a biologically important protective barriers, such as the CD4+ T-cell line), VBL (P-gp overexpressing; selected blood–brain, blood–cerebrospinal fluid, maternal–fetal using vinblastine) and E1000 (MRP-1 overexpress- barriers, testes, and within circulating lymphocytes ing; selected using epirubicin). The expression of these that might be impenetrable to these drugs. To this end, transporters has been previously validated in our lab- because ritonavir-mediated inhibition of cytochrome oratory [13]. The cells were maintained at 37°C, 5% P450 (CYP3A4/CYP2D6) and possibly P-gp has been CO2 in RPMI-1640 media supplemented with 10% shown to increase exposure to coadministered HPIs, fetal bovine serum (FBS). ritonavir (RTV)-boosted HPIs are a standard compo- nent of antiretroviral regimen [28]. Interestingly, we and Isolation of PBMCs others have recently shown that other HPIs, such as ata- PBMCs were isolated from blood buffy coats using Lym- zanavir (ATV), can pharmacoenhance the intracellular phoprep according to the manufacturer’s instructions. accumulation and area under the curve of coadministed HPIs [2,29]. Published evidence to date suggests that Octanol/saline partition coefficient ATV might be a substrate, or induces or inhibits P-gp, As the cellular association of a drug within target cells MRP and BCRP activities [2,3,23,25,30,31], but does is a composite of active influx/efflux, passive diffusion not alter lymphocyte expression of P-gp, and MRP1 and ion trapping, the lipophilicity of [3H]-ATV was [3,31]. The current study aims to characterize the measured as described previously [6] to establish if sim- determinants of the cellular transport and sequestration ple physiochemical characteristics were major determi- of [3H]-ATV in cultured CD4+ T-cells and in primary nants of intracellular accumulation. human lymphocytes – an important site to study the transport and intracellular concentration of antiretro- Transport of [3H]-ATV and the effects of specific virals. Experiments were designed to understand the inhibitors on transport in CEM and CEMVBL cells lipophilicity and sequestration of [3H]-ATV within cells Initial experiments examined the optimal concentrations and the efflux and influx pumps relevant for the vecto- of inhibitors for maximal inhibitory effects on Pgp, MRP rial transport and retention of the drug. and hOATP activity. Cells (CEM and its variant cells) 966 ©2009 International Medical Press AVT-09-OA-1243_Janneh.indd 966 30/10/09 15:14:25 Cellular uptake of atazanavir were incubated in the absence or presence of varying incubating the incubation mixture with 20 µM digitonin concentrations of XR9576 (0.01–1 µM; P-gp/BCRP for 5 min once steady-state was reached (15 min). inhibitor [32–34]), MK571,